Neuronal CCL2 expression drives inflammatory monocyte infiltration into the brain during acute virus infection

Charles L Howe; Reghann G. LaFrance-Corey; Emma N Goddery; Kanish Mirchia

BILs were incubated with 0.5 µg Fc block (anti-FcyRIII/II mAb) prepared from the supernatant of 2.4G2 hybridoma cells for 30 min on ice, followed by staining with CD45 (clone 30-F11, BD Biosciences), CD11b (clone M1/70, BD Biosciences), Ly6G/C (clone RB6-8C5, BD Biosciences), or Ly6G (clone 1A8, BD Biosciences). All antibodies were added to blocked wells at 1:200, incubated for 30 min, and washed three times prior to flow cytometric analysis. Brain infiltrating cells were gated on CD45 expression. All CD45 mid/hi cells were further assessed for expression of CD11b, Ly-6C/G (Gr1), and Ly-6G (1A8). We defined inflammatory monocytes as the CD45 hi CD11b + Gr1 ++ 1A8population, neutrophils as CD45 hi CD11b ++ Gr1 + 1A8 + cells, and microglia as CD45 mid CD11b mid Gr1cells. For phenotyping experiments involving reporter LysM:eGFP mice, we defined inflammatory monocytes as GFP mid cells, neutrophils as GFP hi cells, and microglia as GFP neg cells within a specific forward-and side-scatter gate. Flow cytometric analysis was performed on an Accuri C6 flow cytometer with sampler arm (BD Biosciences, Mountain View, CA). Files were analyzed offline using FlowJo 10.08 (Windows version; FlowJo LLC, Ashland, OR).